Methods for Detecting Isolates of the Potato Virus (Pvy) Responsible for Necroses

ABSTRACT

The invention concerns a method for detecting the PVY virus which consists in a SNP test or an extension reaction specific to mutations corresponding to R/K 400  et D/E 419 , the presence of at least one of said mutations indicating the presence of at least one virulent PVY strain capable of causing necrosis in plants of the Solanaceae family, in particular in potatoes.

The present invention relates to a method for detecting potato virus PVYcomprised of a SNP test or an extension reaction specific for themutations corresponding to R/K₄₀₀ and D/E₄₁₉, the presence of at leastone said mutations being an indication of the presence of a virulentstrain of PVY capable of causing necrosis in plants of the Solanaceaefamily, the potato in particular.

Potato virus Y (PVY), after which the potyvirus group is named, is oneof most important plant pathogens from an economic point of view (Milne,1988; Shukla et al., 1994). First reported in the 1930's in the potato(Smith, 1931), PVY is now distributed throughout the world on a numberof different hosts. The virus is transmitted by aphids in anon-persistent manner (Sigvald, 1984) and infects several species ofcrop plants belonging to the Solanaceae family (De Bokx and Hutting a,1981; Brunt et al., 1996).

The viral genome consists of a single-stranded positive-sense RNAmolecule approximately 10 kb in length, with a VPg protein covalentlybound at its 5′ end and a poly-A tail at its 3′ end. The viral RNA codesfor a polyprotein subsequently cleaved into nine products by threeproteases coded by the virus (Dougherty and Carrington, 1988). Dependingon the host from which they were initially collected, the PVY isolateswere categorized into four different strains, potato, pepper, tobaccoand tomato. Within the potato strain, the isolates were characterized onthe basis of their biological properties (symptoms and responses tovarious sources of resistance). This characterization led to thedefinition of various virus groups. Thus, three groups of potatostrains, PVY^(N), PVY^(O) and PVY^(C) (De Bokx and Hutting a, 1981) wereidentified. These groups are defined by the systemic or local characterof the symptoms induced on Nicotiana tabacum and Solanum tuberosum.

The isolates belonging to the PVY^(N) group induce brown spot disease onthe leaves of N. tabacum cv. Xanthi and a very light mottling, with onlyrarely necrotic leaves in the potato. PVY^(O) isolates induce onlysymptoms of mottling and mosaic on tobacco and a light to serious mosaicand leaf drop in the potato. Finally, PVY^(C) isolates induce symptomsof necrotic streaking in certain potato cultivars.

PVY^(N) and PVY^(O) isolates are responsible for high-yield losses, upto 40% to 70% in the case of the potato. Thus, the efficient detectionand identification of necrotic and non-necrotic PVY isolates in potatocrops are a major problem for producers.

The characterization of PVY “potato” isolates first relied on biologicaltests. However, such an approach requires both time and space and is noteasily adaptable for a rapid diagnosis or for a large-scale test.

Next, to meet the need for a reliable and rapid test, double- ortriple-antibody sandwich enzyme-linked immunosorbent assays (DAS- orTAS-ELISA) using polyclonal and/or monoclonal antibodies (Gugerli andFries, 1983; Matt and Hutting a, 1987; Oshima et al., 1990; Sanz et al.,1990; Singh et al., 1993; Ellis et al., 1996) and an approach byimmunoelectron microscopy (Walkey and Webb, 1984) and by agglutinationwith latex (Berckx, 1967; Tallay et al., 1980) were developed.

Nevertheless, none of these tests proves able to distinguish theisolates capable and incapable of inducing necrosis (Mac Donald andSingh, 1996; Boonham and Barker, 1998; Ounouna, 2002).

Indeed, the recent emergence of new variants of PVY^(N) includingtubular ring necrosis PVY^(NTN) (Le Romancer et al., 1994; Kerlan etal., 1999) and PVY^(N)-W isolates (Chrzanowska, 1991) highlights thelimits of the serological tools available. Indeed, PVY-specificmonoclonal and/or polyclonal antibodies place PVY^(N)-W isolates in thePVY^(O) group. Moreover, serological tools are not able to make thedistinction between PVY^(NTN) and PVY^(N) isolates.

Four complete sequences of PVY^(N) (Robaglia et al., 1989; Jakab et al.,1997; Abdelmaksoud and Gamal Eldin, 2002; Deny and Singh, 2003), twocomplete sequences of PVY^(NTN) (Thole et al., 1993; Deny and Singh,2003) and a complete sequence of PVY^(O) (Singh and Singh, 1996)(reference number: PVYN-Fr: Do00441; PVYN-605: X97895; PVYN-Egypt:AF522296; PVYN-Jg: AY166867; PVYNTN-H: M95491; PVYNTN-Tu660: AY16866;PVYO-139: U09509) were published.

In addition to the serological tools described above, molecular testswere developed by various teams. None of these tools, however, iscapable of precisely characterizing the PVY isolates that inducenecrosis. To solve this problem, we have developed molecular tools thatare faster, more reliable and more specific for the detection of virusescapable of inducing necrosis in an infected plant. In other words, theinvention provides, for the first time, a test enabling suchdiscrimination between various isolates and, more particularly, highlysensitive detection of PVY according to its various biologicalproperties [for example, necroses (Y^(N)) or mottling (Y^(O))] withrespect to the actual biological properties used in the classificationof PVY.

Within the framework of our investigations, we have discovered twomutations in these various isolates directly implicated in necrosis, inparticular tuber necrosis in the potato, and other plants of theSolanaceae family.

This discovery was only made possible by a reverse genetics approach inwhich mutations of amino acids located in the carboxy-terminus portionof the HC-Pro protein were identified as being responsible for necrosis.

The invention now opens the way for the systematic detection of necroticand non-necrotic PVY isolates, which will enable producers tosignificantly decrease the risk of crop loss.

DESCRIPTION

Thus, the present invention relates to a method for detecting thepresence or absence of PVY strains responsible for veinal, foliar ortubercular necrosis in plants of the Solanaceae family, characterizedsuch that it comprises the following steps:

a) extraction of nucleic acids from a plant sample,

b) RT-PCR amplification of a region of PVY viral RNA comprising codons738 and 757 (SEQ ID No 1 and 3), which correspond to amino acids 400 and419, respectively, of the HC-Pro protein (SEQ ID No 2 and 4, 7A and 7B),

c) detecting the presence or absence of the R/K₄₀₀ and D/E₄₁₉ mutations,the detection of at least one said mutation being an indication of avirulent strain of PVY capable of causing necrosis in plants of theSolanaceae family.

In the description, reference will be made to the numbering of NCBIsequence X97895 (Jakab G., Droz E., Brigneti G., Baulcombe D. and MalnoeP., Infectious in vivo and in vitro transcripts from a full-length cDNAclone of PVY-N605, a Swiss necrotic isolate of potato virus, Y. J. Gen.Virol. 78 (Pt 12), 3141-3145 (1997)). The nucleotide and peptidesequences are presented in SEQ ID No 1 and 2, respectively. For PVY^(O)strains, a reference sequence is available at NCBI under number U09509[Singh M. and Singh R. P., Nucleotide sequence and genome organizationof a Canadian isolate of the common strain of potato virus Y (PVYo).Can. J. Plant Pathol. 18, 209-214 (1996)] (SEQ ID NO 3 and 4), but thenumbering will be according to Jakab et al. (1997) by alignment.

In a first embodiment, the invention relates to a method described abovefor detecting the presence or absence of PVY strains responsible forveinal, foliar or tubercular necrosis in plants of the Solanaceaefamily, characterized such that step c) comprises the detection on thecDNA obtained in step b) of the presence or absence of mutationscorresponding to R/K₄₀₀ and D/E₄₁₉ by means of i) at least one labeledprobe specific to a polymorphism on codon 738 and ii) at least onelabeled probe specific to a polymorphism on codon 757, said probes i)and ii) carrying labels that emit a different fluorescent signal, thepresence of at least one of said R/K₄₀₀ and D/E₄₁₉ mutations being anindication of the presence of a virulent strain of PVY responsible fornecrosis in plants of the Solanaceae family.

In a second embodiment, the invention relates to a method describedabove, characterized such that step c) comprises the detection on thecDNA obtained in step b) of the presence or absence of mutationscorresponding to R/K₄₀₀ and D/E₄₁₉ by means of an oligonucleotide primerextension reaction using ddNTP labeled differentially and i) of anunlabeled primer that hybridizes specifically upstream or downstream (±1nt) of polymorphic nucleotide 2213 of codon 738 and ii) of an unlabeledprimer that hybridizes specifically upstream or downstream (±1 nt) ofpolymorphic nucleotide 2271 of codon 757, the presence of at least oneof said R/K₄₀₀ and D/E₄₁₉ mutations being an indication of the presenceof a virulent strain of PVY responsible for necrosis in plants of theSolanaceae family.

Using this method, the following genotypes/phenotypes are detected in asingle test:

-   -   [R₄₀₀, D₄₁₉] (strain incapable of inducing necrosis)    -   [R₄₀₀, E₄₁₉] (strain capable of inducing necrosis)    -   [K₄₀₀, D₄₁₉] (strain capable of inducing necrosis)    -   [K₄₀₀, E₄₁₉] (strain inducing necrosis)

Examples of detected sequences containing one of these combinations ofpolymorphisms are presented in FIG. 6B (SEQ ID No 14 to 19).

As an example, this method can be implemented with the potato. In thiscase, the detection in step c) of PVY [R₄₀₀, E₄₁₉] and/or [K₄₀₀, D₄₁₉]strains and/or [K₄₀₀, E₄₁₉] is an indication that the potato plant iscontaminated with one or more strains capable of inducing tubercularnecrosis (FIG. 6A).

In the first embodiment, the probe i) used in step c) comprises at leastone probe specific to a polymorphism on codon 738 (corresponding toR/K₄₀₀), in particular a probe that hybridizes specifically with thetarget sequence when codon 738 is AAA with polymorphism A₂₂₁₃. The testcan be supplemented with other probes according to other polymorphismson this codon: K (Lysine): AAA, AAG.

Preferably, a probe is used containing from 14 to 40, 15 to 25, 18 to 22or 20 consecutive nucleotides of a sequence capable of hybridizing withsequence SEQ ID NO 5 (FIG. 1) and comprising the nucleotide in position2213, in particular SEQ ID No 7: ctcaaatgaaaatattctac.

A control probe i) can also be used in step c), in particular a probethat hybridizes specifically with the target sequence when codon 738 isAGA with polymorphism G₂₂₁₃. The test can be supplemented with otherprobes according to other polymorphisms on this codon: R (Arginine) CGT,CGC, CGA, CGG, AGA, AGG.

To this end, a control probe can be used containing from 14 to 40, 15 to25, 18 to 22 or 20 consecutive nucleotides of a sequence capable ofhybridizing with sequence SEQ ID No 6 (FIG. 1) and comprising thenucleotide in position 2213, in particular SEQ ID No 8:ctcaaatgagaatattcta.

Advantageously, probe i) and control probe i) are labeled differently.

Also in a preferred embodiment, probe ii) in step c) comprises at leastone probe specific to a polymorphism on codon 757 (corresponding toD/E₄₁₉), in particular a probe that hybridizes specifically with thetarget sequence when codon 757 is GAA with polymorphism A₂₂₇₁. The testcan be supplemented with other probes according to other polymorphismson this codon: E (Glutamic): GAA, GAG.

Preferably, a probe is used containing from 14 to 40, 15 to 25, 18 to 22or 20 consecutive nucleotides of a sequence hybridizing with sequenceSEQ ID NO 5 (FIG. 1) and comprising the nucleotide in position 2271, inparticular with SEQ ID No 9 or SEQ ID No 10, which are specific YNprobes: 5′-cgatcacgaaacgcagaca-3′ (SEQ ID No 9) 5′-atcacgaaacgcagaca-3′(SEQ ID No 20).

A control probe ii) can also be used in step c), in particular a probethat hybridizes specifically with the target sequence when codon 757 isGAC with polymorphism C₂₂₇₁. The test can be supplemented with otherprobes according to other polymorphisms on this codon: D (Aspartic):GAT, GAC.

To this end, a control probe ii) can be used containing from 14 to 40,15 to 25, 18 to 22 or 20 consecutive nucleotides of a sequencehybridizing with sequence SEQ ID No 6 (FIG. 1) and comprising thenucleotide in position 2271, in particular with SEQ ID No 10:5′-accatgacactcaaa-3′ or with SEQ ID No 21: 5′-tgaccatgacactcaa-3′.

Advantageously, probe ii) and control probe ii) are labeled differently.

Probes as described above containing a fluorescent label (reporter) anda molecule capturing the signal when it is near the fluorescent label(quencher) can be used.

In the second embodiment, the method described above is characterizedsuch that the primer i) used in step c) is comprised of at least oneprimer that hybridizes specifically upstream or downstream of thepolymorphic nucleotide of codon 738 (corresponding to R/K₄₀₀), inparticular a primer that hybridizes specifically with the targetsequence when codon 738 is AAA with polymorphism A₂₂₁₃.

Preferably, primer i) contains from 10 to 120 or 20 consecutivenucleotides of a sequence hybridizing with sequence SEQ ID NO 5 (FIG. 1)and comprising the nucleotide in position 2212 or 2214.

In the second embodiment, the primer ii) used in step c) can also be atleast one primer that hybridizes specifically upstream or downstream ofthe polymorphic nucleotide of codon 757 (corresponding to D/E₄₁₉), inparticular a primer that hybridizes specifically with the targetsequence when codon 757 is GAA with polymorphism A₂₂₇₁.

Preferably, primer ii) contains from 10 to 120 or 20 consecutivenucleotides of a sequence hybridizing with sequence SEQ ID NO 5 (FIG. 1)and comprising the nucleotide in position 2270 or 2272.

As examples, in the second embodiment, the detection and identificationof the polymorphic nucleotide in position 2213 is carried out with asense primer selected among:

a sense primer selected among: Oli1: SEQ ID No 345′-GACAACTTGTGCTCAAATGA-3′ Oli2: SEQ ID No 355′-CTGGCGACAACTTGTGCTCAAATGA-3′ Oli3: SEQ ID No 365′-TGGATCTGGCGACAACTTGTGCTCAAATGA-3′ Oli4: SEQ ID No 375′-CATGATGGATCTGGCGACAACTTGTGCTCAAATGA-3′ Oli5: SEQ ID No 385′-CCAACCATGATGGATCTGGCGACAACTTGTGCTCAAATGA-3′ and an antisense primerselected among: Oli6: SEQ ID No 39 5′-GAACATCAGGGTAGAATATT-3′ Oli7: SEQID No 40 5′-ATCATGAACATCAGGGTAGAATATT-3′ Oli8: SEQ ID No 415′-TCTGCATCATGAACATCAGGGTAGAATATT-3′ Oli9: SEQ ID No 425′-GCAGTTCTGCATCATGAACATCAGGGTAGAATATT-3′ Oli10: SEQ ID No 435′-TCTAGGCAGTTCTGCATCATGAACATCAGGGTAGAATATT-3′

In addition, the detection and identification of the polymorphicnucleotide in position 2271 are carried out with a sense primer selectedamong:

Oli11: SEQ ID No 44 5′-GCCTAGAATACTAGTCGATCACGA-3′ Oli12: SEQ ID No 455′-GAACTGCCTAGAATACTAGTCGATCACGA-3′ Oli13: SEQ ID No 465′-GAACTGCCTAGAATATTGGTTGACCATGA-3′ Oli14: SEQ ID No 475′-ATGCAGAACTGCCTAGAATACTAGTCGATCACGA-3′ Oli15: SEQ ID No 485′-ATGCAGAACTGCCTAGAATATTGGTTGACCATGA-3′ and an antisense primerselected among: Oli16: SEQ ID No 49 5′-GTCGACCACATGGCATGTCTGAGT-3′Oli17: SEQ ID No 50 5′-AACGAGTCGACCACATGGCATGTCTGAGT-3′ Oli18: SEQ ID No51 5′-AACGAGTCAACTACATGGCATGTCTGCGT-3′ Oli19: SEQ ID No 525′-AACCAAACGAGTCGACCACATGGCATGTCTGAGT-3′ Oli20: SEQ ID No 535′-AGCCAAACGAGTCAACTACATGGCATGTCTGCGT-3′

Regardless of the embodiment of step c), step b) can be defined asfollows:

The RT and PCR steps take place one after the other in the same tube.For step b), reverse transcription is carried out with at least two orfour pairs of sense and antisense primers, more particularly at least afirst pair enabling the amplification of nucleotide sequences includingcodons 738 and 757 of PVY-N strains, at least a second pair enabling theamplification of nucleotide sequences including codons 738 and 757 ofPVY^(O) strains.

Said first pair of primers (FpN and RpN) preferably comprises an FpNsense primer and an RpN antisense primer that can contain a sequence of20 to 40 consecutive nucleotides of a sequence that hybridizes withsequence SEQ ID No 1 or 5. The FpN sense primer is preferably locatedupstream of polymorphic codon 738 and the RpN primer is preferablylocated downstream of polymorphic codon 757.

Alternately, two first pairs can be used as follows:

-   -   FpNl upstream of codon 738    -   RpNl downstream of codon 738    -   FpN2 upstream of codon 757    -   RpN2 downstream of codon 757

Said second pair of primers (FpO and RpO) preferably comprises an FpOsense primer and an RpO antisense primer that can contain a sequence of20 to 40 consecutive nucleotides of a sequence that hybridizes withsequence SEQ ID NO 3 or 6. The FpO sense primer is preferably locatedupstream of polymorphic codon 738 and the RpO primer is preferablylocated downstream of polymorphic codon 757.

In another alternative, two second pairs can be used as follows:

-   -   FpOl upstream of codon 738    -   RpOl downstream of codon 738    -   FpO2 upstream of codon 757    -   RpO2 downstream of codon 757

This preferred embodiment is not limiting. Indeed, the invention alsorelates to a method such as defined above in which step b) comprises theuse of a number of pairs of primers covering the entire PVY virusspectrum.

An example of an embodiment is presented in FIG. 2. Among these primers,SEQ ID No 11 and 12 can be cited.

In a preferred embodiment, the two pairs of primers are selected among:

Sense primers for YN:

Sense primers for YN: SEQ ID No 22 F1:5′-ATGATGCAGAACTGCCTAGAATACTAGT-3′ SEQ ID No 23 F2:5′-ATGATGCAGAACTGCCTAGAATACTAGTC-3′ SEQ ID No 24 F3:5′-CATGATGCAGAACTGCCTAGAATACTA-3′ Antisense primers for YN: SEQ ID No 28R1: 5′-GTGAGCCAAACGAGTCAACTACAT-3′ SEQ ID No 29 R2:5′-TTTGTGAGCCAAACGAGTCAACTA-3′ SEQ ID No 30 R3:5′-TTGTGAGCCAAACGAGTCAACT-3′ Sense primers for YO: SEQ ID No 25 F1:5′-GCAGAGCTGCCTAGTTTATTGGTT-3′ SEQ ID No 26 F2:5′-ATGATGCAGAGCTGCCTAGTTTATT-3′ SEQ ID No 27 F3:5′-TGCAGAGCTGCCTAGTTTATTGG-3′ Antisense primers for YO: SEQ ID No 31 R1:5′-GCCAAATGAGTCAACCACATGA-3′ SEQ ID No 32 R2:5′-AGCCAAATGAGTCAACCACATG-3′ SEQ ID No 33 R3:5′-CCAAATGAGTCAACCACATGACA-3′

The invention also relates to a sanitary method for selecting seedlingsbelonging to the Solanaceae family contaminated by PVY strainsresponsible for veinal, foliar or tubercular necrosis, in particulartubercular necrosis in the potato, comprising the systematicimplementation of the detection method mentioned above on seeds,seedlings and/or plants to be cultivated and then proceeding with thedestruction or quarantine of said seeds, seedlings or plantscontaminated with a strain exhibiting at least one of the polymorphismscorresponding to:

-   -   [R₄₀₀, E₄₁₉] (strain capable of inducing necrosis)    -   [K₄₀₀, D₄₁₉] (strain capable of inducing necrosis)    -   [K₄₀₀, E₄₁₉] (strain inducing necrosis)

Sanitary control can be carried out as mentioned above with seeds,seedlings and/or plants imported into a given region, for example thoseentering the European Union or Canada or those arriving from riskregions.

In another aspect, the invention relates to a kit for detecting thepresence or absence of PVY viruses responsible for veinal, foliar ortubercular necrosis in plants of the Solanaceae family, characterizedsuch that it comprises:

-   -   at least a first pair of primers enabling the amplification of        nucleotide sequences including codons 738 and 757 of PVY^(N)        strains, at least a second pair of primers enabling the        amplification of nucleotide sequences including codons 738 and        757 of PVY^(O) strains,    -   at least one labeled probe i) specific of a polymorphism on        codon 738 and at least one labeled probe ii) specific for a        polymorphism on codon 757, said probes i) and ii) being such as        described above and carrying labels that emit different        fluorescent signals.

The kit can also include control probes i) and ii) as described above,control probes i) and ii) being labeled with different fluorescentlabels, probes ii) and controls ii) being labeled with differentfluorescent labels.

In the case where the step corresponds to the second embodiment, the kitfor detecting the presence or absence of PVY virus isolates responsiblefor veinal, foliar or tubercular necrosis in plants of the Solanaceaefamily is characterized such that it comprises:

-   -   at least a first pair of primers enabling the amplification of        nucleotide sequences including codons 738 and 757 of PVY^(N)        strains, at least a second pair of primers enabling the        amplification of nucleotide sequences including codons 738 and        757 of PVY^(O) strains,    -   an unlabeled primer that hybridizes specifically upstream or        downstream (±1 nt) from polymorphic nucleotide 2213 of codon 738        and ii) of an unlabeled primer that hybridizes specifically        upstream or downstream (±1 nt) of polymorphic nucleotide 2271 of        codon 757. Numerous examples of sense primers and antisense        primers specific polymorphs 2213 and 2271, respectively, have        been previously described.

The kit can also include a means for extracting the nucleic acids of aplant sample, for example viral RNA crushing buffer and extractionbuffer as described below. The kit can also include the reagentsnecessary for amplification and/or a device for the qualitative andquantitative detection of fluorescent signals.

As an example, the kit can include a solution comprising the primers foruse at an optimal concentration between 400 nM and 1200 nM, inparticular 800 nM, and a solution comprising the probes at aconcentration between 100 nM and 300 nM, in particular 200 nM.

In another aspect, the invention relates to a probe or primer and to theprobe and primer collections mentioned above.

In another aspect, the invention relates to the use of said probes,primers, probe collections and primer collections for detecting thepresence or absence of PVY viruses responsible for veinal, foliar ortubercular necrosis in plants of the Solanaceae family, the potato inparticular, and also eggplant, tomato, pepper or tobacco.

Thus, in the present invention, we propose a nucleotide polymorphismassay for the detection of PVY^(N) and PVY^(O) isolates that targets aspecific molecular label related to the capacity of the members of thePVY^(N) group to cause necrosis. The protocol developed comprises arapid procedure for sampling plants, a rapid nucleic acid extractionprocess (“wet leaf”) and a single-step fluorescent RT-PCR reaction(using at least two TaqMan® probes), which does not require post-PCRhandling.

Such characteristics make it possible to perform up to 96 tests in lessthan three hours, starting with plant sampling and ending with thegeneration of diagnostic results.

The single nucleotide polymorphism (SNP) diagnostic test described abovereliably detected 42 PVY isolates from 13 countries and was able tocorrectly assign them to the PVY^(N) or PVY^(O) group. The samplescontaining more than 10⁴ copies of the PVY^(N) and/or PVY^(O) RNA weredetected efficiently by this test, the latter enabling co-detection ofPVY^(N) and PVY^(O) in mixed infections.

This new detection tool combines the high sensitivity of moleculardetection techniques with speed (RT-PCR series performed in roughly twohours), simplicity (no extraction kit required for sample preparationand a gel-free procedure) and compatibility with robotic apparatusesused for serological assays. The principal improvements provided by thisPVY detection test are, firstly, the choice of using SNP technology,typically used for allelic discrimination assays, genetic segregationanalyses and chromosomal mapping of diploids (for a review, see Oefner,2002).

In addition, we provide for the first time a test that specificallydetects PVY isolates that induce necrosis from a virulent isolates byvirtue of the identification of the characteristics of the polymorphicnucleotides cited above. Applied to haploids (such as single-strandedRNA viruses), the inventive technology has the potential to identifysamples containing only one (visualized as a homozygote) or acombination of two variants (considered by the test as a heterozygote)of the targeted polymorphic sequence.

Thus, the invention also relates to a lot of seeds, seedlings and/orplants of the Solanaceae family, characterized such that it is free ofseeds, seedlings or plants contaminated by PVY and that it exhibits atleast one of the polymorphisms corresponding to:

-   -   [R₄₀₀, E₄₁₉] (strain capable of inducing necrosis)    -   [K₄₀₀, D₄₁₉] (strain capable of inducing necrosis)    -   [K₄₀₀, E₄₁₉] (strain inducing necrosis)

Among these lots, the invention relates to lots of tomatoes, peppers,tobacco, sweet peppers and eggplants. Preferably, the invention relatesto a lot of seedlings or potato tubers, characterized such that that itis free of seedlings or tubers contaminated with a PVY strain and suchthat it exhibits at least one of the polymorphisms corresponding to:

-   -   [R₄₀₀, E₄₁₉] (strain capable of inducing necrosis)    -   [K₄₀₀, D₄₁₉] (strain capable of inducing necrosis)    -   [K₄₀₀, E₄₁₉] (strain inducing necrosis)

The above-mentioned lots are obtained by the implementation of themethod or of the kit described above.

Figure legends will be referred to in the subsequent description.

FIGURE LEGENDS

FIG. 1: Sequences surrounding the polymorphisms responsible for necrosis

FIG. 2: Sequences of PVY^(N)-605 and PVY^(O)-139 used as a target in anucleotide polymorphism assay. The binding sites for the sense primers(FpN and FpO), the antisense primers (RpN and RpO) and the two TaqMan®probes (Probe^(N) and Probe^(O)) are presented. Polymorphic nucleotideA/G₂₂₁₃ is indicated. The specific indicators (reporters) for the probes(FAM and Vic for Probe^(N) and Probe^(O), respectively) are illustratedby a gray square and a circle, respectively. Quenchers, non-fluorescentbinding molecules (MGB [Applied Biosystems]) encorporated at the 3′ endof the fluorescent probes, are indicated by gray stars.^(a) Nucleotidepositions given are as in Jakab et al., (1997).

FIG. 3: Raw fluorescence signal (RFS) obtained using positive (mixedPVY^(N), PVY^(O) and Y^(N)/Y^(O)) and negative (control without matrix)control samples.

A. RFS data for probes specific to PVY^(N) (FAM) and PVY^(O) (Vic). Theaverage values and standard deviations calculated with four replicationsof NTC, pure PVY^(N)-605 and pure PVY^(O)-139 are indicated. The valuesobtained with both replicates of mixed Y^(N)/Y^(o) samples are listed.

B. Schematic representation of RFS data. Each point corresponds to thedata (FAM, Vic) associated with one of the samples tested. Fournon-overlapping regions were defined according to the nature of thesamples tested.

FIG. 4: Schematic representation of the raw (A), target (B) andnormalized (C) fluorescent data. The triangles, squares and circlescorrespond to the pure PVY^(O), NTC and pure PVY^(N) samples. Thetheoretical quantity of PVY RNA present in each sample tested isindicated. FAMt and Vict correspond to the thresholds for effectivedetection of PVY^(N) and PVY^(O), respectively. The regions of detectionand characterization are indicated on the raw (A) and normalized (C)graphs.

FIG. 5: Graphical representation of the co-detection of PVY^(N) andPVY^(O) using SNP fluorescent target data. The detection thresholds forPVY^(N) and PVY^(O) are indicated by FAMt and Vict, respectively. Thesingle detection of PVY^(N) and or PVY^(O) corresponds to regions

and

, respectively. The groups of region

were not detected in the sample. Y^(N)/Y^(O) ratios of 1/100, 1/10,1,10/1 and 100/1 are represented by triangles, circles, squares, starsand diamonds, respectively.

FIG. 6: Analysis of necrotic properties of chimeric PVY isolates in thepotato.

FIG. 7: Sequence PVY^(N)-605, region of interest, HC-PRO

A. Nucleotide sequence (translation initiation codon in bold,polymorphic bases of interest in bold italics)

B. Polyprotein amino acid sequence (containing the underlined HC-Proprotein sequence), the amino acid of interest in bold.

FIG. 8: PVY^(O)-139 sequence, region of interest and HC-PRO

A. Nucleotide sequence (translation initiation codon in bold,polymorphic bases of interest in bold italics)

B. Polyprotein amino acid sequence (containing the underlined HC-Proprotein sequence), amino acid of interest in bold.

FIG. 9: Schematic diagram of the method for characterizing polymorphicnucleotides using the single nucleotide primer extension technique. Thesteps (extraction, RT-PCR, SNaPshot extension and polyacrylamide gelelectrophoresis) ranging from the plant sample to the result obtained bycomputer analysis of the fluorescent signal are presented. Primerslinked to fluorescent ddNTP are visualized using laser excitation aftermigration in a polyacrylamide gel.

FIG. 10: Electropherograms obtained using the technique of labelingspecific primers with ddNTP for the various polymorphic sites to becharacterized. Each site is studied using a specific primer thathybridizes upstream (or downstream; the antisense primer is notillustrated) of the polymorphic site. The various primers have variouslengths in order to enable their characterization for migration. Thepeaks obtained reveal the presence of fluorescent primers in expectedsizes. The nature of the fluorescence enables the characterization ofthe nucleotide of the polymorphic site of interest.

EXAMPLE 1 Origin of PVY Strains and Sample Preparation 1.1 Viruses andHost Plants

Forty-two PVY isolates, characterized serologically and molecularly,belonging to the various PVY groups (PVY^(N) or PVY^(O)) and variants(Y^(NTN) or Y^(N)-W, table 1) were used in this study. PVY^(N)-605(Jakab et al., 1997) and PVY^(O)-139 (Singh and Singh, 1996) were usedas reference isolates for groups PVY^(N) and PVY^(O), respectively. Theywere used to develop the test whereas other PVY isolates were used inthe process of evaluating the assay developed. The isolates weremaintained in a greenhouse by mechanical inoculation on Nicotianatabacum cv. Xanthi, which was used as a test plant in a biological testfor characterizing PVY^(N) and PVY^(O) isolate on the basis of theircapacity or incapacity to induce symptoms of leaf necrosis.

TABLE I Origin of and references for PVY isolates tested within theframework of the invention Strain Isolate Origin Reference N 605Switzerland Jakab et al., 1997 C3VN Scotland Glais et al., 1996 IrlIreland Glais et al., 1996 607 The Netherlands Glais et al., 1996 P21Tunisia Fakhfakh et al., 1996 B203 France Glais et al., 1998 Sp20 SpainBlanco-Urgoiti et al., 1998 Sp125 Spain Blanco-Urgoiti et al., 1998 TVNQCanada Mc Donald and Kristjansson, 1993 B4 France From this report B7France From this report B8 France From this report O 139 Canada Singhand Singh, 1996 Irl Ireland Glais et al., 1998 Sc Scotland Glais et al.,1998 N1702 The Netherlands Glais et al., 1998 Lw Poland Glais et al.,1998 B18 France From this report N-W i-P Poland Glais et al., 1998 N242France Glais et al., 1998 B11 France Glais et al., 2002 Sp17 SpainBlanco-Urgoiti et al., 1998 B15 France From this report N5 France Fromthis report N10 France From this report N12 France From this report N15France From this report N324 France From this report N341 France Fromthis report N362 France From this report NTN Lb Lebanon Glais et al.,1996 FrOrl France Glais et al., 1996 H Hungary Glais et al., 1996 CzLuk1Czech Republic Glais et al., 1996 Sp47 Spain Blanco-Urgoiti et al., 1998Lx2 Tunisia From this report B6 France From this report B9 France Fromthis report N4 France From this report N18 France From this report May2France From this report Dk Denmark From this report

1.2 Sample Preparation

Raw juice was extracted from N. tabacum plants, healthy or infected byPVY, by pressing the leaves (0.5 g) in a cylinder press, in the presenceof 1 ml of cold crushing buffer (PBS; 0.05% (v/v) Tween 20). Thesesamples were used immediately to perform an ELISA or a nucleic acidextraction.

A rapid “wet leaf” extraction procedure adapted from Robert et al.,2000, was performed using three leaf disks (0.2 cm² each, collectedusing a microtube stopper as a perforation device) from each plant. Thematerial collected was incubated for 15 min at 95° C. in 100 μl ofcrushing buffer and then placed at 4° C. for 10 min. Aftercentrifugation (8,000 g for 5 min), the supernatant was collected,transferred to a new tube and diluted 10× in RNase-free water. Extractswere stored at −20° C. until use.

1.3 Immunoenzymatic Assay (ELISA).

PVY was detected in a plant using a double-antibody enzyme-linkedimmunosorbent assay (DAS-ELISA) protocol. Microtiter plate wells werefilled with 1 μg/ml of PVY polyclonal antibody (FNPPPT-INRA, France) ina carbonate buffer (pH 9.6) for 2 h at 37° C. Between each stage of theELISA protocol, the plates were washed three times with PBST buffer(PBS, 0.05% (v/v) Tween 20). 100 μl of raw plant juice were added to thewells which were left until the following day at 4° C. Mouse monoclonalantibodies conjugated with alkaline phosphatase and directed againstPVY^(N) [Bioreba, Switzerland] or PVY^(O/C) [Adgen, UK] were diluted to1/1000 or 1/2000, respectively, in a crushing buffer supplemented with0.2% egg albumin (w/v). According to expected detection specificity, 100μl of these monoclonal antibodies were added to the plate wells for 2hours at 37° C. The plate wells were filled with 100 μl of p-nitrophenylphosphate (1 mg/ml) in a substrate buffer (1 N diethanolamine, pH 9.6)

After incubation for one hour at room temperature, the absorbance of thesamples was read at 405 nm using a microtiter plate reader (TitertekMultiscan [MCC]).

1.4 Preparation of the Standard of Viral RNA for the Test by SNP

Total nucleic acid was extracted from 100 μl of raw juice taken fromplants infected by PVY^(N)-605 or PVY^(O)-139, using a phenol/chloroformprocedure, and suspended in 50 μl of nuclease-free water. Reversetranscription of viral RNA was performed using 3 U of AMV reversetranscriptase [Promega], 10 μmol of the oligonucleotide5′-⁷⁰²GTCTCCTGATTGAAGTTTAC⁹⁶⁸²-3′ (SEQ ID No 13) (nucleotide positionsaccording to isolate PVY^(N)-605), 20 nmol of DNTP, 20 U of RNasin[Promega] and 10 μl of the total nucleic acid extract. The reaction wasperformed according to the enzyme manufacturer's instructions in a finalvolume of 20 μl. The cDNA regions corresponding to part of the HC-Pro/P3genes of PVY^(N)-605 or PVY^(O)-139 were then amplified by PCR using 2.5U of the AmpliTaq polymerase [Applied Biosystems], 40 μmol of theforward primer 5′-aacgtgtttctcgcgatgctaattaacattggcgaggagg-3′ (SEQ ID No11 corresponding to nt 2079 to 2108 of PVY^(O)-139 and comprising anNruI site) and the reverse primer5′-agccatcagtataccaggggataatattgatagaatcaac-3′ (SEQ ID No 12corresponding to nt 2592 to 2561 of PVY^(O)-139, and comprising aBstZ17I site), 20 nmol of dNTP, 75 nmol of MgCl₂ and 10 μl of cDNAadjusted a final volume of 50 μl with sterile water. The reaction wascycled using a Hybaid Express® thermal cycler for 40 cycles at 94° C.for 1 min, 52° C. for 1 min and 72° C. for 1 min. Separately, the PCRproducts corresponding to the PVY^(N) or PVY^(O) sequences were clonedinto the NruI and BstZ17I sites in a modified pBluescript vector(pMTlink), in which the pBluescriptKS [Statagene] multiple cloningcassette was replaced between the KpnI and SacI sites by a shortnucleotide sequence comprising the KpnI-NruI-BstZ17I-SacI uniquerestriction sites. The resulting pMT_(NB) ^(N) and PMT_(NB) ^(O)plasmids were used to produce viral RNA transcription productscorresponding to nucleotides 2086 to 2591 of PVY^(N)-605 andPVY^(O)-139, respectively. Separately 1 μg of pMT_(NB) ^(N) and PMT_(NB)^(O) was linearized by SacI, purified using a phenol/chloroformextraction protocol and suspended in 5 μl of nuclease-free water. PVYRNA transcription products were generated in the presence of 15 U of T3RNA polymerase [Promega], 10 mM of rNTP and 5 mM of dithiotreitol (DTT)for 3 hours at 37° C. The in vitro transcription process wassupplemented by the digestion of the plasmid using RNase-free DNase Ifor 15 min at 37° C. Viral transcription products were extracted using aphenol/chloroform extraction procedure and amyl alcohol, precipitatedand suspended in 100 μl of RNase-free water. Final RNA concentration(μg/μl and copies/μl) was determined by spectrophotometry. PVY^(N) andPWY^(O) in vitro transcription products were diluted in order to obtainsolutions containing 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³ or 10² copies of theviral RNA produced in vitro in 2.5 μl.

EXAMPLE 2 Design of a Primer and a Probe and Detection SNP

PVY nucleotide 2213 (numbered according to Jakab et al., 1997), reportedas being involved in tobacco brown-rib disease from PVY^(N)-605(Balme-Sinibaldi et al., 2004), was chosen to define two probes labeledwith TaqMan®-MGB FAM-(Probe^(N)) or Vic-(Probe^(O)) [Applied Biosystems]corresponding to PVY^(N)-605 or PVY^(O)-139 sequences, respectively(FIGS. 1 and 2). The sense (Fp) and antisense (Rp) primer pairs (FIG. 2)encompassing the sequences targeted by the probe for PVY^(N)-605 (FpNand RpN) and PVY^(O)-139 (FpO and RpO) were designed using PrimerExpress software [Applied Biosystems].

SNP Test Using TaqMan® Fluorescent Probes

TaqMan®-based SNP reactions were carried out in a final volume of 25 μlusing the One-Step RT-PCR Master Mix Reagents Kit [Applied Biosystems]according to the manufacturer's instructions. RT-PCR reactions werecarried out in a single step with 2.5 μl of “wet leaf” extracts fromhealthy or infected plants, or by using transcription products in vitroin the ABI PRISM 7700 Sequence Detection System [Applied Biosystems].Viral RNA reverse transcription was carried out at 48° C. for 30minutes. PCR was performed with a hot-start AmpliTaq polymerase [AppliedBiosystems] using an enzymatic activation step (10 min at 95° C.),followed by denaturation/hybridization/extension cycles (15 sec at 95°C.; 1 min at 60° C.). For each sample, the fluorescent signalscorresponding to FAM, Vic and to the internal control ROX of AppliedBiosystems® were read at the end of the RT-PCR program. The dataobtained (raw fluorescence) were transformed mathematically using SDSvl.7 software [Applied Biosystems] in order to produce datacorresponding to the fluorescent signal of the target component (FAMmand Vicm) and to the normalized fluorescence data (FAMn and Vicn). TheSNP test and the validation procedure were replicated in at least threeindependent experiments.

EXAMPLE 3 Detection of PVY^(N)-605 and PVY^(O)-139 in Pure and MixedSamples Using an SNP Assay Following Example 2

According to the manufacturer's recommendations, and taking into accountpreviously published real-time RT-PCR protocols (Fabre et al., 2003;Roberts et al., 2000), the two TaqMan® probes (Probe^(N) and Probe^(O))and the primer pairs (FpN and RpN; FpO and RpO) were tested at variousconcentrations from 50 nM to 900 M. The fluorescent signals collected atthe end of the PCR reactions were optimal when each probe was includedat 200 nM and the four primers at 800 nM. Thirty-two PCR cycles wereperformed in all of the detection experiments in order to avoid thenonspecific probe cleavage observed in experiments using more than 35PCR cycles. The SNP test was repeated four times, including a controlwithout a matrix (NTC) or in vitro viral RNA transcription products (10⁶PVY^(N) or 10⁶ PVY^(O) copies/reaction) and with duplication of themixed samples containing PVY^(N) and PVY^(O) (10⁶ of each type of RNA).The raw fluorescence signals (RFS) associated with each probe wererecorded at the end of the single-step RT-PCR reaction (FIG. 3). For theNTC samples, RFS corresponds to the baseline system fluorescence level(0.782±0.024 and 0.424±0.008 for the fluorescence of FAM and Vic,respectively), produced by the uncleaved probes (FIG. 3A). When thesamples containing RNA transcription products were tested, theprobe-associated RFS increased according to the type of RNA present inthe samples tested [Probe^(N) (FAM signal) and Probe^(O) (Vic signal)for PVY^(N) and PVY^(O) respectively] This result shows highly specifichybridization of the probes to their RNA targets and the absence of anysignificant level of nonspecific interaction. In the mixed Y^(N)/Y^(O)samples, the fluorescent signals of both FAM and Vic increasedsignificantly when compared with NTC data, reflecting the binding andcleaving of two probes for the PCR reaction. The graphicalrepresentation of raw fluorescent signal data (FIG. 3B) illustrates boththe distinction of four regions corresponding to each sample tested andthe variation in the fluorescence level within these regions recordedduring each replication.

EXAMPLE 4 Test of the Sensitivity of the PVY^(N/O) SNP Test

Fractions from a series of dilutions containing from 10⁷ to 10³ copiesof RNA/2.5 μl of in vitro PVY^(N) and PVY^(O) transcripts were producedand tested using the SNP test protocol in order to establish thedetection limit of this novel method (FIG. 4). Good correlation could beobserved between decreasing viral quantity in the samples tested and thedrop in fluorescent signal, for both PVY^(N and PVY) ^(O) (FIG. 4A). Thethree most concentrated dilutions were effectively detected andidentified as PVY^(N) or PVY^(O) by SNP assay (FIG. 4A); 10⁷, 10⁶ and10⁵ fractions in the PVY^(N) and PVY^(O) regions). However, when thesamples containing only 10⁴ and 10³ RNA molecules were tested, thefluorescent data obtained were either close to (10⁴) orindistinguishable from (10³) those associated with NTC samples. Thesedata variations for the NTC replications (FIGS. 3A and 3B) indicate thatit is difficult to clearly identify the fluorescent threshold thatdelimits PVY-positive detection from PVY-negative detection. In order tosolve this problem, the SDS® software [Applied Biosystems] enablesmathematical transformations of the raw fluorescent signal into targetfluorescence data (FIG. 4B) and normalized fluorescence data (FIG. 5C).These data make it possible to precisely set the SNP assay evaluationcriterion dilution in the range of 10⁴ to 10⁵ for PVY^(N) and PVY^(O).

EXAMPLE 5 Co-Detection of Mixed PVY^(N) and PVY^(O) in Samples Mimickinga Co-Infection

By using fractions containing between 10⁴ and 10⁸ PVY^(N) or PVY^(O) invitro transcription products [NruI-BsZ17I], several mixed fractions wereprepared by creating samples with Y^(N)/Y^(O) ratios of 1/100 to 100/1(table 2 and FIG. 5). These fractions were tested as unknown samples inan SNP assay comprising NTC, pure PVY^(N) control samples (106transcription products) and pure PVY^(O) (10⁶ transcription products).The raw fluorescence data obtained from the pure samples (FAM=1.753 andVic=1.276 for PVY^(N) and PVY^(O), respectively) were equivalent tothose obtained in the mixed samples containing 106 copies of the twotypes of RNA (1/1 ratio; 10 copies of PVY^(N) and PVY^(O); FAM=1.653 andVic=1.198). This illustrates that single detection and co-detection ofPVY^(N) and/or PVY^(O) in samples containing similar quantities oftargeted RNA were effective for both targets. As previously shown, thequantity of PVY RNA present in the samples tested influences the rawfluorescence level directly.

Fractions containing 108 copies of both types of PVY RNA were associatedwith high RFS values (close to 2.000), whereas the fractions containingonly 10⁴ copies of PVY RNA produced RFS data (FAM=0.860 and Vic=0.563)close to those expected for the negative control (NTC; FAM=0.754 andVic=0.427). The use of target fluorescence data (table 2) with the FAMtand Vict detection threshold (FIG. 5) made it easy to distinguish singledetection from co-detection. By taking the observations into account,the fractions with quantities of PVY RNA from 10⁵ to 10⁸ with aY^(N)/Y^(O) ratio of 1 were effectively characterized as mixed samples.When fractions containing one type of PVY RNA in excess (ratio=1/100,1/10, 10/1 or 100/1) were tested, the RFS for a constant quantity of atype of PVY RNA is reduced according to the excess of the other type ofPVY RNA (PVY^(N)=10⁶: FAMm was 1.216, 0.970, 0.798, 0.454 and 0.68 forthe samples containing 10⁴, 10⁵, 10⁶, 10⁷ and 10⁸ PVY^(O),respectively).

EXAMPLE 6 Validation of the SNP Test on a Wide Range of PVY Isolates

The SNP test developed was validated using a wide range of PVY isolatescomprising 37 European isolates, two African isolates, twoNorth-American isolates and one isolate from the Near East, belonging tothe PVY^(N) and PVY^(O) groups, comprising the variants PVY^(NTN) andPVY^(N)-W (table 1). The biological and serological properties of 42isolates were characterized using observations of symptoms on N. tabacumcv. Xanthi and ELISAs (table 3). All of the results were in agreementwith the expected results. “Wet leaf” extracts of N. tabacum cv. Xanthiinfected by these PVY isolates were prepared and then tested using theSNP test developed. The target fluorescence data and the SNP diagnosticresults associated with each sample tested are presented in table 3. All42 of the PVY isolates tested could be correctly assigned by the SNPtest to their respective PVY group. Variants PVY^(NTN) and PVY^(N)-Wwere correctly characterized as members of the PVY^(N) group. As wasexpected, none the samples tested from our collection of PVY isolateswas identified by the SNP test as a co-infected sample.

EXAMPLE 7 Specific Detection of PVY^(N) and PVY^(O) Isolates Using aFluorescent Nucleotide (ddNTP) Primer Extension Technique

After having extracted the nucleic acids from the plant to be tested, anRT-PCR step is carried in a way similar to that described in example1.4. The amplification product obtained is then purified using a step offiltration on a Sephadex G-50 matrix in order to eliminate the freenucleotides and the components (enzyme, primers, etc.) still present inthe sample. The purified PCR product (amplicon) then undergoes anextension step via a cyclic polymerization reaction (10 seconds at 96°C., 5 seconds at 50° C. and 30 seconds at 60° C.) performed 25 times inthe presence of the primers that hybridize upstream or downstream of thepolymorphic sites to be characterized and a Taq polymerase enzyme in thepresence of ddNTP (having a differential fluorescent marking). At theend of this single extension phase, the samples are placed in thepresence of one unit of alkaline phosphatase and incubated for one hourat 37° C. in order to limit the disturbance to the reading of theresults by free ddNTP. The enzyme then is decontaminated by incubationof the sample for 15 minutes at 75° C. Lastly, the sample is depositedon a polyacrylamide gel and migrates under the effect of an electricfield. In order to be able at the end to position the variousfluorescent signals recorded during migration, a size marker (standardpossessing fluorescent nucleotide fragments of various sizes from 10 to120 nucleotides) was added to each sample before being deposited on theelectrophoresis gel. Polyacrylamide gel migration rate beingproportional to molecule size (length), the size marker makes itpossible to identify the size of each primer observed during migration.The nature of the fluorescence of these primers makes it possible toidentify the nature of the fluorescent ddNTP to which it is linked. ThisddNTP fluorescence corresponds to the complementary base of thepolymorphic nucleotide initially present on the target molecule presentin the sample tested. The general principles of this test are diagrammedin FIGS. 9 and 10.

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1. A method for detecting the presence or absence of PVY strainsresponsible for veinal, foliar or tubercular necrosis in plants of theSolanaceae family, characterized such that it comprises the followingsteps: a) extraction of nucleic acids from a plant sample, b) RT-PCRamplification of a region of PVY viral RNA comprising codons 738 and 757(SEQ ID No 1 and 3), which correspond to amino acids 400 and 419,respectively, of the HC-Pro protein (SEQ ID No 2 and 4, 7A and 7B), c)detecting the presence or absence of the R/K₄₀₀ and D/E₄₁₉ mutations,the detection of at least one said mutation being an indication of avirulent strain of PVY capable of causing necrosis in plants of theSolanaceae family.
 2. A method according to claim 1, characterized suchthat step c) comprises the detection on the cDNA obtained in step b) ofthe presence or absence of mutations corresponding to R/K₄₀₀ and D/E₄₁₉by means of i) at least one labeled probe specific to a polymorphism oncodon 738 and ii) at least one labeled probe specific to a polymorphismon codon 757, said probes i) and ii) carrying labels that emit adifferent fluorescent signal, characterized such that the presence of atleast one of said R/K₄₀₀ and D/E₄₁₉ mutations is an indication of thepresence of a virulent strain of PVY responsible for necrosis in plantsof the Solanaceae family.
 3. A method according to claim 1,characterized such that step c) comprises the detection on the cDNAobtained in step b) of the presence or absence of mutationscorresponding to R/K₄₀₀ and D/E₄₁₉ by means of an oligonucleotide primerextension reaction using ddNTP labeled differentially and i) of anunlabeled primer that hybridizes specifically upstream or downstream (±1nt) of polymorphic nucleotide 2213 of codon 738 and ii) of an unlabeledprimer that hybridizes specifically upstream or downstream (±1 nt) ofpolymorphic nucleotide 2271 of codon 757, the presence of at least oneof said R/K₄₀₀ and D/E₄₁₉ mutations being an indication of the presenceof a virulent strain of PVY responsible for necrosis in plants of theSolanaceae family.
 4. A method according to claim 1, characterized suchthat in a single test the 4 following genotypes/phenotypes are detected:[R₄₀₀, D₄₁₉] (strain incapable of inducing necrosis) [R₄₀₀, E₄₁₉](strain capable of inducing necrosis) [K₄₀₀, D₄₁₉] (strain capable ofinducing necrosis) [K₄₀₀, E₄₁₉] (strain inducing necrosis)
 5. A methodaccording to claim 1, characterized such that it is implemented in thepotato.
 6. A method according to claim 2, characterized such that thedetection in step c) of PVY [R₄₀₀, E₄₁₉] and/or [K₄₀₀, D₄₁₉] strainsand/or [K₄₀₀, E₄₁₉] is an indication that the potato plant iscontaminated with one or more strains capable of inducing tubercularnecrosis.
 7. A method according to claim 2, characterized such that theprobe i) used in step c) comprises at least one probe specific to apolymorphism on codon 738 (corresponding to R/K₄₀₀), in particular aprobe that hybridizes specifically with the target sequence when codon738 is AAA with polymorphism A₂₂₁₃.
 8. A method according to claim 7,characterized such that probe i) contains from 14 to 40, 15 to 25, 18 to22 or 20 consecutive nucleotides of a sequence capable of hybridizingwith sequence SEQ ID NO 5 (FIG. 1) and comprising the nucleotide inposition 2213, in particular SEQ ID No 7: ctcaaatgaaaatattctac.
 9. Amethod according to claim 7, characterized such that in addition acontrol probe i) is used in step c), in particular a probe thathybridizes specifically with the target sequence when codon 738 is AGAwith polymorphism G₂₂₁₃.
 10. A method according to claim 9,characterized such that control probe i) contains from 14 to 40, 15 to25, 18 to 22 or 20 consecutive nucleotides of a sequence capable ofhybridizing with sequence SEQ ID No 6 (FIG. 1) and comprising thenucleotide in position 2213, in particular SEQ ID No 8:ctcaaatgagaatattcta.
 11. A method according to claim 9, characterizedsuch that probe i) and control probe i) are labeled differently.
 12. Amethod according to claim 2, characterized such that a probe ii) is usedin step c) comprised of at least one probe specific to a polymorphism oncodon 757 (corresponding to D/E₄₁₉), in particular a probe thathybridizes specifically with the target sequence when codon 757 is GAAwith polymorphism A₂₂₇₁.
 13. A method according to claim 12,characterized such that probe ii) contains from 14 to 40, 15 to 25, 18to 22 or 20 consecutive nucleotides of a sequence hybridizing withsequence SEQ ID NO 5 (FIG. 1) and comprising the nucleotide in position2271, in particular with SEQ: 5′-cgatcacgaaacgcagaca-3′ (SEQ ID No 9) or5′-atcacgaaacgcagaca-3′ (SEQ ID No 20).
 14. A method according to claim12, characterized such that in addition a control probe ii) is used instep c), in particular a probe that hybridizes specifically with thetarget sequence when codon 757 is GAC with polymorphism C₂₂₇₁.
 15. Amethod according to claim 14, characterized such that control probe ii)contains from 14 to 40, 15 to 25, 18 to 22 or 20 consecutive nucleotidesof a sequence hybridizing with sequence SEQ ID No 6 (FIG. 1) andcomprising the nucleotide in position 2271, in particular with SEQ ID No10: 5′-accatgacactcaaa-3′ or with SEQ ID No 21: 5′-tgaccatgacactcaa-3′.16. A method according to claim 14, characterized such that probe ii)and control probe ii) are labeled differently.
 17. A method according toclaim 7, characterized such that the probes contain a fluorescent label(reporter) and a molecule that captures the signal when near thefluorescent label (quencher).
 18. A method according to claim 3,characterized such that the primer i) used in step c) is comprised of atleast one primer that hybridizes specifically upstream or downstream ofthe polymorphic nucleotide of codon 738 (corresponding to R/K₄₀₀), inparticular a primer that hybridizes specifically with the targetsequence when codon 738 is AAA with polymorphism A₂₂₁₃.
 19. A methodaccording to claim 18, characterized such that primer i) contains from10 to 120 or 20 consecutive nucleotides of a sequence hybridizing withsequence SEQ ID NO 5 (FIG. 1) and comprising the nucleotide in position2212 or
 2214. 20. A method according to claim 3, characterized such thatthe primer ii) used in step c) is at least one primer that hybridizesspecifically upstream or downstream of the polymorphic nucleotide ofcodon 757 (corresponding to D/E₄₁₉), in particular a primer thathybridizes specifically with the target sequence when codon 757 is GAAwith polymorphism A₂₂₇₁.
 21. A method according to claim 20,characterized such that primer ii) contains from 10 to 120 or 20consecutive nucleotides of a sequence hybridizing with sequence SEQ IDNO 5 (FIG. 1) and comprising the nucleotide in position 2270 or 2272.22. A method according to claim 1, characterized such that in step b),reverse transcription is carried out with at least two pairs of senseand antisense primers.
 23. A method according to claim 22, characterizedsuch that reverse transcription is carried out with at least two or fourpairs of sense and antisense primers, more particularly at least a firstpair enabling the amplification of nucleotide sequences including codons738 and 757 of PVY^(N) strains, at least a second pair enabling theamplification of nucleotide sequences including codons 738 and 757 ofPVY^(O) strains.
 24. A method according to claim 23, characterized suchthat the first pair of primers (FpN and RpN) preferably comprises an FpNsense primer and an RpN antisense primer that can contain a sequence of20 to 40 consecutive nucleotides of a sequence that hybridizes withsequence SEQ ID No 1 or
 5. 25. A method according to claim 23,characterized such that the FpN sense primer is located upstream ofpolymorphic codon 738 and the RpN primer is located downstream ofpolymorphic codon
 757. 26. A method according to claim 23, characterizedsuch that two first pairs can be used as follows: FpNl upstream of codon738 RpNl downstream of codon 738 FpN2 upstream of codon 757 RpN2downstream of codon 757
 27. A method according to claim 23,characterized such that the second pair of primers (FpO and RpO)preferably comprises an FpO sense primer and an RpO antisense primerthat can contain a sequence of 20 to 40 consecutive nucleotides of asequence that hybridizes with sequence SEQ ID NO 3 or
 6. 28. A methodaccording to claim 27, characterized such that the FpO sense primer islocated upstream of polymorphic codon 738 and the RpO primer is locateddownstream of polymorphic codon
 757. 29. A method according to claim 27,characterized such that two second pairs are used as follows: FpOlupstream of codon 738 RpOl downstream of codon 738 Fp02 upstream ofcodon 757 Rp02 downstream of codon 757
 30. A method according to claim23, characterized such that the two pairs of primers are selected among:Sense primers for YN: Sense primers for YN: SEQ ID No 22 F1:5′-ATGATGCAGAACTGCCTAGAATACTAGT-3′ SEQ ID No 23 F2:5′-ATGATGCAGAACTGCCTAGAATACTAGTC-3′ SEQ ID No 24 F3:5′-CATGATGCAGAACTGCCTAGAATACTA-3′ Antisense primers for YN: SEQ ID No 28R1: 5′-GTGAGCCAAACGAGTCAACTACAT-3′ SEQ ID No 29 R2:5′-TTTGTGAGCCAAACGAGTCAACTA-3′ SEQ ID No 30 R3:5′-TTGTGAGCCAAACGAGTCAACT-3′ Sense primers for YO: SEQ ID No 25 F1:5′-GCAGAGCTGCCTAGTTTATTGGTT-3′ SEQ ID No 26 F2:5′-ATGATGCAGAGCTGCCTAGTTTATT-3′ SEQ ID No 27 F3:5′-TGCAGAGCTGCCTAGTTTATTGG-3′ Antisense primers for YO: SEQ ID No 31 R1:5′-GCCAAATGAGTCAACCACATGA-3′ SEQ ID No 32 R2:5′-AGCCAAATGAGTCAACCACATG-3′ SEQ ID No 33 R3:5′-CCAAATGAGTCAACCACATGACA-3′


31. A method according to claim 3, characterized such that the detectionand identification of the polymorphic nucleotide in position 2213 iscarried out with sense primer selected among: Oli1: SEQ ID No 345′-GACAACTTGTGCTCAAATGA-3′ Oli2: SEQ ID No 355′-CTGGCGACAACTTGTGCTCAAATGA-3′ Oli3: SEQ ID No 365′-TGGATCTGGCGACAACTTGTGCTCAAATGA-3′ Oli4: SEQ ID No 375′-CATGATGGATCTGGCGACAACTTGTGCTCAAATGA-3′ Oli5: SEQ ID No 385′-CCAACCATGATGGATCTGGCGACAACTTGTGCTCAAATGA-3′

and an antisense primer selected among: Oli6: SEQ ID No 395′-GAACATCAGGGTAGAATATT-3′ Oli7: SEQ ID No 405′-ATCATGAACATCAGGGTAGAATATT-3′ Oli8: SEQ ID No 415′-TCTGCATCATGAACATCAGGGTAGAATATT-3′ Oli9: SEQ ID No 425′-GCAGTTCTGCATCATGAACATCAGGGTAGAATATT-3′ Oli10: SEQ ID No 435′-TCTAGGCAGTTCTGCATCATGAACATCAGGGTAGAATATT-3′


32. A method according to claim 3, characterized such that the detectionand identification of the polymorphic nucleotide in position 2271 arecarried out with a sense primer selected among: SEQ ID No 44 Oli11:5′-GCCTAGAATACTAGTCGATCACGA-3′ SEQ ID No 45 Oli12:5′-GAACTGCCTAGAATACTAGTCGATCACGA-3′ SEQ ID No 46 Oli13:5′-GAACTGCCTAGAATATTGGTTGACCATGA-3′ SEQ ID No 47 Oli14:5′-ATGCAGAACTGCCTAGAATACTAGTCGATCACGA-3′ SEQ ID No 48 Oli15:5′-ATGCAGAACTGCCTAGAATATTGGTTGACCATGA-3′

and an antisense primer selected among: SEQ ID No 49 Oli16:5′-GTCGACCACATGGCATGTCTGAGT-3′ SEQ ID No 50 Oli17:5′-AACGAGTCGACCACATGGCATGTCTGAGT-3′ SEQ ID No 51 Oli18: 5′-AACGAGTCAACTACATGGCATGTCTGCGT-3′ SEQ ID No 52 Oli19: 5′-AACCAAACGAGTCGACCACATGGCATGTCTGAGT-3′ SEQ ID No 53 Oli20:5′-AGCCAAACGAGTCAACTACATGGCATGTCTGCGT-3′


33. A sanitary method for selecting seedlings belonging to theSolanaceae family contaminated by PVY strains responsible for veinal,foliar or tubercular necrosis, in particular tubercular necrosis in thepotato, comprising the systematic implementation of the detection methodaccording to claim 1 on seeds, seedlings and/or plants to be cultivatedand then proceeding with the destruction or quarantine of said seeds,seedlings or plants contaminated with a strain exhibiting at least oneof the polymorphisms corresponding to: [R₄₀₀, E₄₁₉] (strain capable ofinducing necrosis) [K₄₀₀, D₄₁₉] (strain capable of inducing necrosis)[K₄₀₀, E₄₁₉] (strain inducing necrosis).
 34. A kit for detecting thepresence or absence of PVY viruses responsible for veinal, foliar ortubercular necrosis in plants of the Solanaceae family, characterizedsuch that it comprises: at least a first pair of primers enabling theamplification of nucleotide sequences including codons 738 and 757 ofPVY^(N) strains, at least a second pair of primers enabling theamplification of nucleotide sequences including codons 738 and 757 ofPVY^(O) strains, at least one labeled probe i) specific of apolymorphism on codon 738 and at least one labeled probe ii) specificfor a polymorphism on codon 757, said probes i) and ii) being such asdescribed according to claim 5, respectively and carrying labels thatemit different fluorescent signals.
 35. A detection kit according toclaim 34 comprising in addition control probes i) and ii) as definedaccording to claim 7, respectively, probes i) and controls i) beinglabeled with different fluorescent labels, probes ii) and controls ii)being labeled with different fluorescent labels.
 36. A kit for detectingthe presence or absence of PVY virus isolates responsible for veinal,foliar or tubercular necrosis in plants of the Solanaceae family ischaracterized such that it comprises: at least a first pair of primersenabling the amplification of nucleotide sequences including codons 738and 757 of PVY^(N) strains, at least a second pair of primers enablingthe amplification of nucleotide sequences including codons 738 and 757of PVY^(O) strains, an unlabeled primer that hybridizes specificallyupstream or downstream (±1 nt) from polymorphic nucleotide 2213 of codon738 and ii) of an unlabeled primer that hybridizes specifically upstreamor downstream (±1 nt) of polymorphic nucleotide 2271 of codon
 757. 37. Adetection kit according to claim 34, that includes a solution comprisingthe primers for use at an optimal concentration between 400 nM and 1200nM, in particular 800 nM, and a solution comprising the probes at aconcentration between 100 nM and 300 nM, in particular 200 nM.
 38. Aprobe or probe collection defined according to claim
 2. 39. A primer orprimer collection defined according to claim
 3. 40. The use of a probeor probe collection according to claim 38 and/or of a primer or primercollection according to claim 39 for detecting the presence or absenceof PVY strains responsible for veinal, foliar or tubercular necrosis inplants of the Solanaceae family, in particular in eggplant, potato,tomato, pepper and tobacco.
 41. A lot of seeds, seedlings and/or plantsof the Solanaceae family, characterized such that it is free of seeds,seedlings or plants contaminated by PVY and that it exhibits at leastone of the polymorphisms corresponding to: [R₄₀₀, E₄₁₉] (strain capableof inducing necrosis) [K₄₀₀, D₄₁₉] (strain capable of inducing necrosis)[K₄₀₀, E₄₁₉] (strain inducing necrosis).
 42. A lot of seedlings orpotato tubers, characterized such that that it is free of seedlings ortubers contaminated with a PVY strain and such that it exhibits at leastone of the polymorphisms corresponding to: [R₄₀₀, E₄₁₉] (strain capableof inducing necrosis) [K₄₀₀, D₄₁₉] (strain capable of inducing necrosis)[K₄₀₀, E₄₁₉] (strain inducing necrosis).
 43. A lot according to claim 41obtained by the implementation of the method according to claim 33.